The present invention concerns a process for inhibiting cyclic nucleotide independent protein kinase activity and related tumor cell growth.
Protein kinases represent a class of cellular enzymes which function, in a broad sense, as activators or modulators of other cellular proteins. One subclass of protein kinases, termed cyclic nucleotide dependent, requires the presence of cyclic AMP or cyclic GMP for activity. These proteins have been implicated in many aspects of normal cell regulation, and typically phosphorylate target proteins at serine or threonine residues.
A second subclass, termed cyclic nucleotide independent protein kinases, phosphorylate proteins at tyrosine and serine residues. However, tyrosine phosphorylation accounts for only about 0.05% of total protein phosphate in normal cells. By comparison, cellular phosphoserine and phosphothreonine are, collectively, 3000 times more abundant. Sefton, et al., Cell 20:807 (1980).
Possible roles for tyrosine phoshorylation in cellular regulation and virus-associated malignant cell transformation have been reviewed by Hunter, TIBS 7:246 (1982) and Erikson, et al., J. Cell. Biol 87:319 (1980). These authors suggest a link between cell transformation by tumor viruses and the appearance of enhanced tyrosine-specific protein kinase activity. Although increased levels of phosphotyrosine have not been observed for all transforming viruses, at least ten viruses have been associated with this phenomenon. For example, chick fibroblasts transformed by Rous sarcoma virus (RSV) exhibit 6-10 fold increases in cellular phosphotyrosine.
The role and ontogeny of tyrosine phosphorylation in cell transformation by certain tumor viruses is unclear. Tyrosine phosphorylation has been implicated in control of normal cell growth processes involving epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin receptor. See Kasuga et al. Proc. Nat. Acad. Sci, USA 80:2137 (1983); Cohen et al., J. Bio. Chem. 255:4834 (1980); and Sefton et al., supra. Erikson et al., J. Biol. Chem. 256:11381 (1981) describe experiments in which protein kinases observed in normal cells stimulated by EGF, and in tumor cells transformed by RSV, were shown to phosphorylate an identical cellular protein with similar specificity. Thus, it has been theorized that inappropriately elevated levels of tyrosine-phosphorylating activity may contribute to neoplastic growth of transformed cells through phosphorylation of normal growth receptors.
In a search for effective antineoplastic agents, several workers have tested certain chloromethyl ketones and diazomethyl ketones as possible inhibitors of transformation-specific protein kinase activity. Richert et al., Cell 18:369 (1979), disclose experiments in which N-.alpha.-tosyl-L-lysyl chloromethyl ketone (TLCK) was observed to inhibit in vivo protein phosphorylation activity associated with pp60.sup.src, an avian sarcoma virus (ASV) gene product linked to fibroblast transformation. Weber, Cell 5:253 (1975) had previously reported phenotypic reversion of ASV-transformed cells to normal upon treatment with TLCK.
Johnson et al., Biochemistry 21:2984 (1982) describe inhibition of hexokinase and protein kinase activities of Ehrlich ascites tumor cells by a chloromethyl ketone derivative of lactic acid, 2-isobutyl 3-oxo-4-chloro-2-butyl carbonate. Antitumor activity has also been described for certain N-tosyl amino acid diazomethylketones and chloromethyl ketones by Sajadi et al., J. Med. Chem. 23:275 (1980). Navarro, et al., Biochemistry 21:6138 (1982), disclose inhibition of tyrosine protein kinase activity in certain tumor cell preparations by halomethyl ketone derivatives of amino acids and peptides.
Thus, new methods of inhibiting cyclic nucleotide independent protein kinase activity are of interest to the medical community as new approaches to treatment and control of neoplastic cell growth.